Presentation: 2025 ND EPSCoR Annual conference 

October 21, 2025, NDSU Memorial Union, Fargo, North Dakota

Characterization of Two Single Chain “Calmodulin – Calmodulin Binding Peptide” Constructs

Calmodulin (CaM) is a critically important mediator of intracellular calcium (Ca2+) signaling and interacts with a large number of Ca2+/CaM regulated proteins. A key mechanism of CaM regulation of its target is the binding of CaM in the presence of Ca2+ to specific peptide sequences known as CaM-binding peptides (CaMBP). Because multiple binding modes between CaM and its binding peptides have been documented, it is important to experimentally characterize the mode of CaM binding to each target. The pseudokinase CAMKV contains a predicted CaM binding peptide. We explore a new approach to define the binding between CaM and the CAMKV derived CaMBP and to gain structural information about the orientation of CAMKV CaMBP when bound to CaM. We have engineered two single chain constructs in with the CAMKV-CaMBP is fused to the N- or C-terminus of CaM via a flexible linker. We present data on the expression, isolation and purification of both constructs. The biophysical characterization includes circular dichroism spectroscopy, fluorescence spectroscopy, dynamic light scattering and electrophoretic mobility in the presence and absence of Ca2+. Attempts to crystallize the CAMKV-CaMBP protein for X-ray diffraction experiments are ongoing.